Subtercola endophyticus sp. nov., a cold-adapted bacterium isolated from Abies koreana

A novel Gram-stain-positive, aerobic bacterial strain, designated AK-R2A1-2 T, was isolated from the surface-sterilized needle leaves of an Abies koreana tree. Strain AK-R2A1-2 T had 97.3% and 96.7% 16S rRNA gene sequence similarities with Subtercola boreus K300T and Subtercola lobariae 9583bT, respectively, but formed a distinct phyletic lineage from these two strains. Growth of strain AK-R2A1-2 T was observed at 4–25 °C at pH 5.0–8.0. Strain AK-R2A1-2 T contained menaquinone 9 (MK-9) and menaquinone 10 (MK-10) as the predominant respiratory quinones. The major cellular fatty acids were anteiso-C15:0 and summed feature 8 (C18:1ω7c or/and C18:1ω6c), and the polar lipids included diphosphatidylglycerol (DPG) and three unknown aminolipids, AKL2, AKL3, and AKL4. The complete genome of strain AK-R2A1-2 T was sequenced to understand the genetic basis of its survival at low temperatures. Multiple copies of cold-associated genes involved in cold-active chaperon, stress response, and DNA repair supported survival of the strain at low temperatures. Strain AK-R2A1-2 T was also able to significantly improve rice seedling growth under low temperatures. Thus, this strain represents a novel species of the genus Subtercola, and the proposed name is Subtercola endophyticus sp. nov. The type strain is AK-R2A1-2 T (= KCTC 49721 T = GDMCC 1.2921 T).

Bootstrap values (> 50%) were calculated using the NJ, maximum-likelihood (ML), and minimum evolution (ME) algorithms. Filled circles on the nodes indicate that the relationships were also identified using the ML and ME algorithms, whereas open circles indicate nodes were identified by either the ML or the ME algorithm. Scale bar: 0.0100 substitutions per nucleotide position. www.nature.com/scientificreports/ Phenotypic characteristics. Phenotypic characteristics based on the optimal growth medium, temperature, pH, and salt tolerance were investigated. Strain AK-R2A1-2 T was shown to grow well on R2A, PDA, MEA, YEP, ISP2, and GYM medium, but did not on MA, LB, NA, and TSA. Cells were short and rod-shaped without flagella (0.2-0.3 µm in width and 0.3-1.6 µm in length, Fig. S1), and were Gram stain-positive, non-motile, catalase-positive, and oxidase-negative. Growth was observed in R2A medium at 4-25 °C (optimal 20 °C), pH 5-8 (optimal pH 5), and 0-1% NaCl (optimal 0%). Differential physiological characteristics that distinguish strain AK-R2A1-2 T from closely related strains are shown in Table 1.

Genome analyses. General and functional features of the AK-R2A1-2 T genome.
After assembly using the Canu (version 1.7) de novo assembler, the whole genome of strain AK-R2A1-2 T was shown to comprise a single circular chromosome of 4,318,731 bp, and the N50 value was 12,176 bases, with coverage of 95x. The G + C content calculated based on the respective whole-genome sequence was 65.8%. A comparison between two copies of the 16S rRNA gene fragments with the whole-genome sequence showed that DNA sequence contamination did not occur during the genome assembly of strain AK-R2A1-2 T . The whole genome sequence of the novel strain was deposited into NCBI under accession number CP087997.1. The genome sequence was annotated using RAST 9,10 , and protein-coding sequences were determined using PGAP 11 . The NCBI PGAP annotation revealed that the AK-R2A1-2 T genome contained 3,874 protein-coding genes and 56 RNA genes, including two 5S rRNA, two 16S rRNA, two 23S rRNA, three non-coding RNA (ncRNA), and 47 tRNA genes (Table S1). A total of 3,674 protein-coding sequences (CDSs) were annotated using the cluster orthologous group (Fig. 2). The largest group of CDSs was classified as unknown (1,106 of the total CDSs, 30.1%), while the groups with the lowest CDSs numbers were classified as cytoskeleton and RNA processing and modification (1 of the total CDSs, 0.02%). Transcription (8.9%) and carbohydrate transport and metabolism (7.8%) gene had the next highest numbers of CDSs annotated from the whole genome ( Fig. 2 and S3). A whole genome-based phylogenetic tree was reconstructed using UBCG (version 3.0) and showed that strain AK-R2A1-2 T forms a subgroup with S. lobariae 9583b T , and that all members of the genus Subtercola comprise one group (Fig. 3). Concurrently, members of the genus Agreia form a single cluster, consistent with the phylogenetic tree created using 16S rRNA gene sequences. These findings suggested that strain AK-R2A1-2 T was a novel species of the genus Subtercola.
Genomic features associated with the cold-adaption of strain AK-R2A1-2 T . The RAST server, PGAP, and Blast-KOALA pipeline were used to perform genome annotation and determine the metabolic pathways of strain AK-R2A1-2 T . The following information is predicted from the genome sequence analyses: Stress response Members of the genus Subtercola grow well at low temperatures, and while strain AK-R2A1-2 T was isolated at 25 °C, it was shown to grow at 4 °C; the type strains of other Subtercola members can even grow at − 2°C 5 . The potential mechanism(s) by which bacteria invoke stress responses to adapt to environmental changes have not yet been fully elucidated. Bacterial adaptations to facilitate survival at low temperatures include cell membrane fluidity (increased unsaturated fatty acids) and protein-specific responses (refolding cold-damaged proteins) 12 . Cold-shock proteins (Csp) are produced by bacteria in response to a rapid decrease in temperature and act as RNA chaperones to help prevent mRNA misfolding 13 . The PGAP analysis in the current study showed that the AK-R2A1-2 T genome contained two genes encoding a cold-shock protein (UFS59601.1; UFS60376.1) and a cold shock domain-containing protein (UFS59609.1). When whole-genome mining of strain AK-R2A1-2 T was performed using RAST SEED and BlastKOALA, many well-studied, cold-inducible genes 14 , rbfA (ribosome-binding factor A), recA (recombination protein RecA), and tig (trigger factor) ( Table 3). Compatible solutes, a group of compounds that are important for osmotic stress and cold shock, can reduce membrane stress using various mechanisms. Biosynthetic genes of the recognized coldprotective solutes, glycine betaine (opuC, opuBD, opuA), choline (betaA), and proline (proA, proB, proC) were annotated 21,22 . In addition, genes responsible for the synthesis of the osmoprotectant proline from glutamate
Using the RAST SEED annotation of strain AK-R2A1-2 T (Fig. S4), 22 genes were associated with the stress response. Of these, three were involved in osmotic stress (osmoregulation), 11 were related to oxidative stress [oxidative stress (6), glutathione: redox cycle (3), glutaredoxins (1), glutathionylspermidine and trypanothione  www.nature.com/scientificreports/ (1)], and eight were related to detoxification. Some freezing-protective solutes, which can contribute to cold stress remission 28,29 , such as amino sugar and nucleotide sugars (26), amino acids (96), starch, and sucrose (27) were also detected in the genome information from strain AK-R2A1-2 T . Motility Bacterial motility is critical to its establishment in and colonization of its host, and promotes plant growth 30 . Two genes related to motility, one for flagellar assembly (rpoD) and one for bacterial chemotaxis (rbsB), were annotated from the whole genome of strain AK-R2A1-2 T . The incomplete motility pathway of strain AK-R2A1-2 T explains why it was non-motile.
Fatty acid metabolism Membrane fatty acids are the major determinants for a sufficiently fluid membrane state required for low-temperature membrane function. The melting point of membrane fatty acids at cold temperatures is reduced by the synthesis of short-chain fatty acids, increases in the relative abundance of mono/ polyunsaturated fatty acids, and increases in the anteiso/iso branched-chain fatty acids ratio [31][32][33][34] 35 , was detected in the whole genome of strain AK-R2A1-2 T . In addition, fatty acid biosynthesis genes, fabD, fabF, fabG, fabH, fabL, OXSM, OAR1, and unsaturated fatty acid biosynthesis-related genes including tesB were detected in the whole genome.
Genome comparison and biosynthetic potential of the novel strain. The proposed threshold values of dDDH and ANI for delineating novel bacterial species are < 70% and < 95-96%, respectively 36 . In the current study, the ANI values were 76.8-80% between strain AK-R2A1-2 T and other members of the genus Subtercola, 73.6-74% between AK-R2A1-2 T and members of the genus Agreia, and < 74.7% between AK-R2A1-2 T and other genera in the family Microbacteriaceae. Consistently, strain AK-R2A1-2 T exhibited a dDDH value of 22.5-24.5% with members of the genus Subtercola, 20.1-20.5% with members of the genus Agreia, and < 20.7% with other genera in the family Microbacteriaceae (Table S2). Similar results were obtained for orthoANI values, which were < 80.3% with most closely related strains in the family Microbacteriaceae (Table S2).
Bacteria produce a large number of secondary metabolites that act as a reservoir of bioactive metabolites, with some exhibiting unique functions such as salt resistance 37    www.nature.com/scientificreports/ on the production of specialized metabolites and the potential source of natural products remains untapped. In recent years, many computational methods have been developed to identify biosynthetic gene clusters (BGCs) in genomic data. AntiSMASH is a platform that is widely used for genome mining of secondary metabolites. The secondary metabolite clusters of the AK-R2A1-2 T genome were searched using antiSMASH (V. 6.0.1). Seven clusters were identified from the whole genome and found to contain non-alpha poly-amino acids like ε-polylysine, microansamycin (polyketide), alkylresorcinol (polyketide), kosinostatin (NPR + polyketide), beta-lactone, carotenoid (terpene), and lipopolysaccharide (saccharide: lipopolysaccharide) (Table S3 and Figs. S5-S11). Some of these compounds have vital antioxidant, antimicrobial, and anti-inflammatory properties. For example, NAPAA is a cationic peptide shown to prevent microbe proliferation and is approved as a foodgrade cationic antimicrobial metabolite 38 , which has natural antioxidant and antimicrobial activities 39 . Carotenoid can be used to protect against oxidative stress in model systems [40][41][42][43] , and regulates membrane fluidity at low temperature using cell membrane adaptation 44,45 . Higher carotenoid levels may also increase resistance to cold shock 46 . Alkylresorcinol can protect epithelial cells against oxidative damage and has antioxidant 47-49 , antigenotoxic 49 and potential antiglioma activity 50 . Kosinostatin has antitumor, antimicrobial, and antiproliferative functions 51,52 , while beta-lactone is a natural product with significant clinical applications provided by its antimicrobial, anticancer, and anti-obesity properties 53,54 . Microansamycin is a member of the macrolactams family, important bioactive compounds with anti-tuberculous and anti-tumor activity 55 . Similarly, its host, the Korean fir, produces rich polyphenol compounds like quercetin (flavonoid) and carotenoids to provide the strong antioxidant properties of Korean fir 56 . The essential oil of Korean fir includes the terpene group (monoterpenes, oxygenated monoterpenes, sesquiterpenes, diterpenes, and limonene) 57,58 which also have strong antioxidant, antimicrobial, and anti-inflammatory activities 1,4 . The functional secondary metabolites of strain AK-R2A1-2 T were very similar to its host plant.

Effect of strain AK-R2A1-2 T on rice seedling growth under low temperature. Many endophytic
bacteria have a broad host range. For example, Bacillus altitudinis, isolated from the wild plant species, Glyceria chinensis, can promote the growth of Arabidopsis thaliana, N. tabacum, corn, and soybean 59 . Bacillus megaterium RmBm31, an endophytic bacterium isolated from Retama monosperma, increases the biomass of A. thaliana 60 . Rice is a cold-sensitive crop, and its exposure to low-temperature stress (< 20 °C) during germination and early seedling growth, can have a negative impact on initial stand establishment 61,62 . To assess the impact of AK-R2A1-2 T on rice seedling growth under low temperatures, rice seeds were co-cultivated with strain AK-R2A1-2 T for 10 days at 20 °C, and total fresh weight, shoot weight, shoot length, root number, root length, and chlorophyll contents were examined in the rice seedling (Fig. 4). Shoot weight and shoot length were 1.67fold and 1.36-fold higher, respectively, in the AK-R2A1-2 T -exposed seedlings than in controls (CK), while root www.nature.com/scientificreports/ length and root number were 1.42-fold and 1.40-fold higher, respectively, in the AK-R2A1-2 T -exposed seedings (Fig. 4). Strain AK-R2A1-2 T significantly improved rice seedling biomass and root morphological parameters under low temperatures. Whole genome analysis showed that these improvements were associated with phytohormone biosynthesis genes for indole-3-acetic acid (trpABCDES) and phosphate solubilization (pstABCS, phoHLU, and phnB). Indole-3-acetic acid can induce the growth of auxin-dependent lateral root formation, root hair development, and primary root growth, which promote plant growth. Meanwhile, phosphate-solubilizing bacteria enhance plant production by solubilizing insoluble phosphorus to make it available and increasing phosphorus nutrition. Secondary metabolites including terpenes were also shown to promote plant growth and some BGCs may produce new secondary metabolites because they display low similarity to known clusters. The function of these secondary metabolites on plant growth will require further study.

Description of
The GenBank accession numbers of the 16S rRNA gene and whole genome sequences of strain AK-R2A1-2 T are OL314543 and CP087997.1, respectively. The strain is available from the Korea Collection for Type Culture (KCTC 49721 T ) and the Guangdong Microbial Culture Collection Center (GDMCC 1.2921 T ).

Materials and methods
Sample isolation and culture conditions. Plant samples were collected from the top region of Mount Halla, Jeju Island, Republic of Korea (33°21′32.4″ N, 126°31′68″ E) in July 2020. Two grams of needle leaves from Abies koreana tree samples were surface-sterilized with 1% sodium hypochlorite, rinsed five times in sterile distilled water, and crushed with 20 mL of 1 × phosphate-buffered saline (PBS) in a grinder. Ground samples were serially diluted from 10 −1 to 10 −4 with 1 × PBS, and 100 µL aliquots were spread onto tenfold diluted Reasoner's 2A agar (R2A, Difco), and incubated at 25 °C for 7 days. Morphologically distinct colonies were selected and subsequently streaked on fresh R2A medium. All strains were preserved in sterile skimmed milk (10%, w/v) at − 80 °C. Among all the isolates, one white, circular, convex, and smooth colony, designated AK-R2A1-2 T , was selected for further study. Subtercola boreus DSM 13056 T (=K300 T ), Subtercola vilae DSM 105013 T (=DB165 T ), Subtercola frigoramans KCTC 49696 T (=K265 T ), and Subtercola lobariae KCTC 33586 T (=9583b T ) were obtained from the corresponding culture collections as closely related strains to analyze the taxonomic characteristics under comparable culture conditions. Unless otherwise stated, bacterial strains were grown on R2A or ISP2 for 5 days for the subsequent tests.
Phenotypic characteristics. AK-R2A1-2 T cell morphology was observed by scanning electron microscopy at the Chuncheon Center, Korea Basic Science Institute (KBSI) after growing the strain on R2A agar plates for 4 days. Gram staining was conducted with a Gram staining kit (Difco) according to the manufacturer's instructions. Cell motility was tested by growing the strain on a semi-solid R2A medium (0.4%). Catalase activity was determined by the production of bubbles after adding 3% (v/v) hydrogen peroxide solution to fresh cells 64 , while oxidase activity was measured using oxidase test strips (bioMérieux) that changed color to purple. Different media including R2A, potato dextrose agar (PDA, Difco), malt extract agar (MEA, Difco), yeast extract peptone agar (YEP, Difco), ISP medium No.2 (ISP2, Difco), and GYM medium (GYM: glucose 4 g/L, yeast extract 4.0 g/L, malt extract 10 g/L, CaCO 3 2 g/L, agar 15 g/L), marine agar 2216 (MA, Difco), Luria-Bertani agar (LB, Difco), nutrient agar (NA, Difco), and trypticase soy agar (TSA, Difco) were screened for optimal growth of the tested strains. Various temperatures (4,10,15,20,25,30,37,40,45, and 60 °C) were assayed to identify the growth range of the novel strain. The pH range (3.0 to 12.0, adjusted with 1 N HCl and NaOH) for growth and NaCl tolerance (0 to 15%, w/v; 1% concentration increments) were measured in R2A liquid medium, monitoring the optical density at 600 nm (OD 600 ) using a microplate spectrophotometer (Multiskan skyhigh, Thermo Fisher Scientific). Other biochemical features, such as the utilization of substrates, acid production from carbohydrates, and enzyme activities, were tested using API 20NE (bioMérieux) or API ZYM with www.nature.com/scientificreports/ NaCl 0.85% medium (bioMérieux) and API 50CH according to the manufacturer's protocols. All closely related type strains were tested under the same conditions. Chemotaxonomy features. Biomass for analysis of cellular fatty acids from strain AK-R2A1-2 T and closely related type strains was extracted from cells grown in R2A media. Using the Sherlock Microbial Identification System version 6.0 (MIDI) standard protocol, whole-cell fatty acid methyl esters were extracted and analyzed by gas chromatography (model 6890 N; Agilent) using the Microbial Identification software package 65 . Quinones were extracted from 100 mg of freeze-dried cells by shaking in chloroform: methanol (2:1, v/v) and purified by thin-layer chromatography as described by Collins et al. 66 . Analysis of the purified quinones was performed using reverse-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection at 270 nm. For extraction and analysis of polar lipids, 100 mg of freeze-dried cells were boiled in methanol, mixed and shaken with chloroform, evaporated, and identified by two-dimensional thin-layer chromatography (TLC) on Kieselgel 60 F254 plates (silica gel, 10 × 10 cm; Merck). Lipids spots were visualized by spraying with 0.2% ninhydrin (Sigma-Aldrich), α-naphthol, molybdenum blue (Sigma-Aldrich), 4% phosphomolybdic acid reagent, and Dragendorff 's solution to detect amino group-containing, sugar-containing, phosphorus-containing, total, and quaternary nitrogen-containing lipids, respectively.
Genome sequencing and assembly. Whole genome sequencing was performed by the Macrogen facility (Macrogen, Republic of Korea) using the PacBio Sequel System (Pacific Biosciences, Inc.) and the Illumina sequencing platform. Raw sequencing data were assembled using the Canu (version 1.7) de novo assembler 67 . Error corrections were performed by Pilon (version 1.21) 68 . Potential contamination of the assembled genome was checked using the ContEst16S algorithm 69 to compare 16S rRNA within the whole genome. To verify that strain AK-R2A1-2 T belonged to the genus Subtercola, a whole genome-based phylogenetic tree was reconstructed using the up-to-date bacterial core gene set and pipeline (UBCG) as described by Na et al. 70 .
Genome annotation and comparative analysis. The National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotation of microbial genomes using Subsystem Technology (RAST) SEED 9-11 were used for genome annotation. Metabolic pathways were reconstructed using BlastKOALA, which is based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database 71 . Genome mining for the presence of secondary metabolite gene clusters was performed using the antiSMASH program (https:// antis mash. secon darym etabo lites. org/) with a "relaxed detection strictness" parameter and a "known cluster blast" feature including non-ribosomal peptide synthetases (NRPSs), type I and type II polyketide synthases (PKSs), lanthipeptides, lasso peptides, sactipeptides, and thiopeptides 72 . Comparative genome analyses including digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) values, and orthoANI values between strain AK-R2A1-2 T and the most closely related members of the family Microbacteriaceae were performed using the Genome-to-Genome Distance Calculation (GGDC) webserver (http:// ggdc. dsmz. de/) 73 , ANI calculator (https:// www. ezbio cloud. net/), and standalone Orthologous Average Nucleotide Identity (OAT) software 74 , respectively. A graphical circular map of the AK-R2A1-2 T genome was constructed using CGView (https:// cgview. ca/). Rice growth and low-temperature conditions. Seeds of the Japonica rice cultivar Haepung were surface-sterilized with 1% (v/v) sodium hypochlorite for 10 min followed by repetitive washes with distilled water and soaked in AK-R2A1-2 T suspension (1 × 10 8 CFU/mL) or R2A broth as a control as described as Jiang et al. 37 .
The control and AK-R2A1-2 T bacteria-coated seeds were incubated in the dark at 14 °C for 4 days in three replicated square plates (245 mm × 245 mm) containing 0.15% water agar. All the plates were incubated at 20 °C with a 16 h light/8 h dark photoperiod for 10 days. To estimate shoot weight, shoot length, root weight, root number, and root length, fifteen seedling plants from each experiment were considered. Chlorophyll contents were calculated as described by Fu et al. 75 . All experiments were performed in triplicate yielding similar results.

Data availability
The strain is available from the Korean Collection for Type Cultures (KCTC 49721 T ) and the Guangdong Microbial Culture Collection Center (GDMCC 1.2921 T ). The GenBank accession number of AK-R2A1-2 T for the 16S rRNA gene is OL314543. The whole genome sequence of AK-R2A1-2 T accession number is CP087997.1. The associated BioSample and BioProject accession numbers are SAMN23075623 and PRJNA678113, respectively. The taxonomy ID is 2,895,559.